Behavior-linked FoxP2 regulation enables zebra finch vocal learning

Abstract

Mutations in the FOXP2 transcription factor cause an inherited speech and language disorder, but how FoxP2 contributes to learning of these vocal communication signals remains unclear. FoxP2 is enriched in corticostriatal circuits of both human and songbird brains. Experimental knockdown of this enrichment in song control neurons of the zebra finch basal ganglia impairs tutor song imitation, indicating that adequate FoxP2 levels are necessary for normal vocal learning. In unmanipulated birds, vocal practice acutely downregulates FoxP2, leading to increased vocal variability and dynamic regulation of FoxP2 target genes. To determine whether this behavioral regulation is important for song learning, here, we used viral-driven overexpression of FoxP2 to counteract its downregulation. This manipulation disrupted the acute effects of song practice on vocal variability and caused inaccurate song imitation. Together, these findings indicate that dynamic behavior-linked regulation of FoxP2, rather than absolute levels, is critical for vocal learning.

Keywords: basal ganglia; birdsong; forkhead; language; procedural learning; speech.

Figure 1.

Exogenous overexpression of speech-related FoxP2 during zebra finch vocal learning. a, Schematic depicts control (GFP) and FoxP2-expressing viral constructs delivered by stereotaxic injection into song-dedicated Area X. b, Top, Coronal hemisections illustrate targeting and expression in Area X, visible in the Nissl stain (left), and indicated by the dashed line (right). In situ hybridization signals for zebra finch FoxP2 reveal elevated mRNA at the injection site of the FoxP2-expressing virus. Bottom, Dense, restricted GFP expression at bilateral injection sites of the control virus. c, Mid- (left) and high- (middle) power images of the brain shown in b reveals overlap between viral-driven GFP-expression (green) and NeuN immunostain (red). Venn diagram (right) illustrates the quantitative overlap (yellow) between GFP and NeuN. d, Comparison of viral-driven GFP expression (AAV1-GFP) and immunostain signals for endogenous FoxP2 (Endog FoxP2) indicate high levels of overlap (Merged). e, Representative immunoblot of FoxP2 signals arising from Area X micropunches in an adult bird that was injected with AAV1-GFP in one hemisphere and AAV1-FoxP2 contralaterally. An overall 40.3% increase in FoxP2 signal was observed in hemispheres receiving the AAV-FoxP2 virus relative to the contralateral side (p = 0.0436, n = 7 pairs, paired one-tailed bootstrap). f, Timeline of behavioral experiments.

Figure 2.

FoxP2 overexpression during the sensorimotor critical period disrupts vocal learning. a, Spectrograms (frequency range of 0–11 kHz) depict motifs from a tutor and his three pupils which each received a stereotaxic injection of AAV1 driving either GFP expression (GFP) or FoxP2 (FoxP2+). Scale bars, 200 ms. Syllables that correspond across motifs are underlined with black bars and identified by letters (question marks indicate unidentifiable syllables). b, Quantification of the similarity of each pupil's motif to its tutor reveals that FoxP2+ birds (gray bars) have lower scores than those of GFP birds (green bars; p = 0.0269, n = 8GFP/10FoxP2+, unpaired one-tailed bootstrap). Midline represents mean, upper and lower bounds of the box represent SE, upper and lower whiskers represent 95% confidence intervals, and points represent individual birds. c, Manual counting of syllables revealed that FoxP2 overexpression leads to an increase in the number of tutor syllables that were omitted by the pupil (p = 0.0247, n = 8GFP/10FoxP2+, unpaired one-tailed bootstrap). In contrast, GFP and FoxP2+ pupils exhibit similar levels of improvised syllables (p = 0.3040, n = 8GFP/10FoxP2+, unpaired one-tailed bootstrap). d, The motifs of GFP and FoxP2+ pupils exhibit similar levels of syntax similarity to their tutor's motif (p = 0.2276, n = 7GFP/8FoxP2+, unpaired one-tailed bootstrap). e, Exemplar spectrograms of a different tutor and his three pupils (1 GFP, 2 FoxP2+) highlight the low fidelity imitation of tutor syllables by FoxP2+ pupils. f, Poor syllable imitation by FoxP2+ relative to GFP pupils is reflected in lower-syllable identity scores (p = 0.0067, n = 8GFP/10FoxP2+, unpaired one-tailed bootstrap). g, Syllable-by-syllable comparison of the feature-specific errors made by FoxP2+ pupils versus their GFP sibling. Black points above unity represent syllables for which the FoxP2+ sibling made larger errors, whereas green points below unity represent syllables for which the GFP sibling made larger errors. FoxP2+ pupils made larger errors for duration (100-sequential match), FM entropy, and goodness, but not pitch nor AM (p = 0.0115, 0.0004, 0.0239, 0.0108, 0.1196, 0.0761, respectively; n = 41 syllables, paired one-tailed bootstrap).

Figure 3.

FoxP2 overexpression leads to imitation deficits that emerge early despite similar developmental trajectories. a, Spectrograms depict representative motifs of two pupils (GFP control and FoxP2+) at 3 stages of sensorimotor learning (50, 70, and 90 d) and that of their shared tutor. b, Motif identity scores indicate the similarity of a pupil's motif to that of its tutor. Scores are plotted for three ages of each control GFP (green) and FoxP2+ (black) pupil. The latter group had lower scores at 50 d (p = 0.0028, n = 5GFP/8FoxP2+, unpaired one-tailed bootstrap), 70 d (p = 0.0398, n = 7GFP/7FoxP2+, unpaired one-tailed bootstrap), and 90 d (p = 0.0082, n = 8GFP/8FoxP2+, unpaired one-tailed bootstrap). The motifs of both groups of pupils became increasingly similar to that of the tutor between 50 and 70 d (GFP: p = 0.0471, n = 5; FoxP2+: p = 0.0166, n = 7; paired one-tailed bootstrap), but not between 70 and 90 d (GFP, p = 0.2772, n = 7; FoxP2+: p = 0.1786, n = 7; paired one-tailed bootstrap). c, Motif identity scores indicate the similarity of a pupil's motif to its own adult version. Scores are plotted for three ages of each control GFP (green) and FoxP2+ (black) pupil. Both groups followed similar developmental trajectories manifested by increases in similarity to adult song between 50 and 70 d (GFP: p = 0.0204, n = 5; FoxP2+: p = 0.0214, n = 8; paired one-tailed bootstrap), and between 70 and 90 d (GFP: p = 0.0004, n = 7; FoxP2+: p = 0.0214, n = 7; paired one-tailed bootstrap) and no difference between groups at the 50, 70, or 90 d time points (p = 0.7517, 0.6381, 0.6366, respectively; unpaired one-tailed bootstrap).

Figure 4.

FoxP2 overexpression does not affect variability at adulthood. Green and gray bars represent GFP controls and FOXP2+ pupils, respectively. a, Overall rendition-to-rendition variability of syllables, as measured by syllable identity, was not significantly different between groups (p = 0.8489, n = 8GFP/10FoxP2+, unpaired two-tailed bootstrap). b, FoxP2 overexpression did not affect feature-specific variability, as measured by the coefficient of variability for duration, amplitude, pitch, FM, entropy, PG, mean frequency (p = 0.2685, 0.5548, 0.5703, 0.7217, 0.8237, 0.928, 0.8371, respectively; n = 8GFP/10FoxP2+, unpaired two-tailed bootstrap). c, FoxP2 overexpression did not affect syntax variability (p = 0.8489, n = 7GFP/8FoxP2+, unpaired two-tailed bootstrap).

Figure 5.

FoxP2 overexpression disrupts behavior-dependent transitions between low and high variability during learning. a, The approach and hypotheses are schematized. On adjacent days, birds were prevented from singing for 2 h or were allowed to sing undirected song for 2 h. The vocal variability immediately following these two epochs was measured. We predicted transitions between variability states in the GFP birds but not FoxP2+ birds. b, Exemplar syllables are shown here with their individual measurements and entropy CV and self-identity measurements based on 20 renditions of the syllable. c, We found divergent effects of FoxP2 overexpression on feature-specific variability, exemplified here by PG, pitch, and entropy CV. In each example, GFP birds showed significantly elevated variability following vocal practice (UD-UD). In FoxP2+ birds, however, the effect of vocal practice depended on the feature being measured: there was no effect on PG CVs, an increase in pitch CV in UD-UD condition, and a decrease in entropy CV in the UD-UD condition. The net result of these changes is a global practice induced increase in variability in GFP birds, which is blocked in FoxP2+ birds. d, A summary diagram of all the feature-specific and global changes observed. Notably, in GFP birds the semicoordinated practice-induced change in variability across multiple features gives rise to a global increase in variability. In contrast, in FoxP2+ birds feature-specific changes are not coordinated and do not give rise to a global change in variability. The means, confidence intervals, and p values represented here are shown in Table 1.